Enterotoxigenic Escherichia coli have been implicated as causative agents in travelers diarrhea, infantile diarrhea and colibacillosis in infant animals and they have been shown to elaborate a cholera-like heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin (ST). LT activates adenylate cyclase to induce excess intestinal secretion. ST activates guanylate cyclase to result in the same physiological effect. ST has been purified, and has been shown to be a small peptide, containing 18 amino acids. The mechanism by which it activates guanylate cyclase in intestinal mucosa is unknown and that is the focus of this project. We will use pig intestinal mucosa, and isolate the brush-border membranes from the enterocytes. It is our hypothesis that the binding of ST to its receptor in the membrane, induces activation of guanylate cyclase by a mechanism analogous to the better described hormonal activation of adenylate cyclase. That activation is mediated by membrane proteins shown to bind GTP, and GTP is required for the transmembrane signalling to occur. The quanylate cyclase system differs in that GTP is the substrate for the enzyme, hence a GTP requirement for guanylate cyclase activation has heretofore been missed. We will investigate this system by first studying the binding of (125I) to its membrane receptor as affected by GTP and its analogues. Then we will study the binding of labeled GTP and analogues ST to the membranes and attempt to photoaffinity label GTP-binding proteins. We will attempt to discover if hemin, which inhibits ST activation, does so by interacting with a coupling protein. Finally, we will purify and characterize the GTP-binding proteins from these membranes.